Adsorption and binding properties of enzymes are important in controlling their retention in, or leaching from, films formed of polyelectrolytes such as polyanions and polycations. They are also important in controlling their binding to matrices, from which they may be eluted, or to which they may be anchored, and in controlling heir adsorption on surfaces to which they may be well attached, or from which they may be readily removed by rinsing. They are also relevant to controlling their reversible or irreversible binding with biological macromolecules, such as nucleic acids, and with non-biological macromolecules, whether in a homogeneous solution, or when immobilized on a surface. Controlled binding to, or expulsion from, surfaces is particularly relevant to assaying biochemicals and immunochemicals. By changing the adsorption or binding characteristics, one can improve selectivity, signal to noise ratio and signal stability in these assays.
The charge of an enzyme affects its adsorption on surfaces, absorption in films, electrophoretic deposition on electrode surfaces, and interaction with macromolecules when the surfaces, films or macromolecules contain covalently or coordinatively bound cations or anions. When the concentration of dissolved small ions, that have charges of an opposite sign to that of the enzyme at a given pH, is not particularly high, then electrostatic repulsion reduces adsorption on, absorption in, or binding to surfaces, films or macromolecules, when the charges of these and the charge of the enzyme are of the same sign. When their charges and the charge of the enzyme are of opposite signs, adsorption, absorption or binding are enhanced. It is, therefore, of importance in diagnostic and analytical systems utilizing enzymes to tailor the charge of the enzyme so as to enhance its adsorption onto a surface, its absorption or retention in a film, or its binding to a macromolecule that may be dissolved in a solution or in a film on a surface. In other cases the opposite is desirable, i.e. it is important to facilitate desorption, removal, or stripping of the enzyme from a surface or matrix by making its interaction with a surface, or dissolved or immobilized macromolecule, repulsive. For example, in films used in optical or electrochemical blood glucose analyzers, it is desirable to retain the enzyme glucose oxidase (GOx) or the enzyme recombinant glucose oxidase (rGOx) in a film. Furthermore, it is preferred that the film contain an anionic polymer, termed polyanion, because polyanions partially exclude anionic species that interfere with the assays, such as ascorbate or urate. The unmodified enzymes GOx and rGOx, that in the physiological range (7.3.+-.0.7) are polyanions, are better retained in polycationic films. These interact, however, with anionic interferants attractively. The attractive interaction increases the rate of permeation of interferant through the film. This enhances the undesired interference by the interferant. If the enzyme is, however, converted into a cationic or positively charged macromolecule, termed a polycation, then the enzyme is better retained in polyanionic films, from which interferants are partially excluded.
Control of the binding or repulsion of an enzyme from a surface is particularly important in immunoassays. These depend on specific binding of antigens and antibodies and on the prevention of non-specific binding to other surfaces or macromolecules. Most immunoassays involve the use of marker-enzymes that are chemically, usually covalently, bound to either an antigen or to an antibody. In immunoassays it is important to prevent non-specific binding of the marker-enzyme. Such non-specific binding can be reduced by using as the marker an enzyme that, because of its charge, i.e. its being a polyanion or a polycation, repulsively interacts with a surface or macromolecule involved in the immunoassay, thus assuring that the dominant attractive interaction is that between the specific antigen and the specific antibody. Here control of the charge of the enzyme enhances the specificity of the method, e.g. of enzyme linked immunosorbent assay referred to as ELISA.
Enzymes are also used to mark nucleic acids, e.g. DNA. They are used to label sequences of nucleotides and thus determine the presence or absence of complementary sequences, or to quantify the amount of specific sequences in test samples. These tests are based on highly specific interactions of components of genetic information carrying matter. In these, the enzyme, that labels or marks a sequence of nucleotides, must not itself bind non-selectively to the nucleic acid that is being probed with the enzyme labeled nucleotide sequence. By making the interaction between the nucleic acid that is probed and the enzyme electrostatically repulsive, non-specific binding can be avoided.